The tumor suppressor p53 as an ideal therapeutic target for gliomas. The results of our recent phase I clinical trial (PI: F.F. Lang) of adenovirus-p53 gene delivery (Ad-p53), and those of other studies in our laboratory, demonstrate that the anticancer effect of Ad-p53 occurs via apoptosis. The data also show that, whereas glioma cells harboring mutant p53 undergo apoptosis following Ad-p53 infection, wild-type p53 glioma cells are resistant to Ad-p53-induced apoptosis. Treatment of wild-type p53 glioma cells with DNA damaging agents after Ad-p53 infection results in significant apoptosis. Recent findings from our laboratory indicate that phosphorylation of p53 may be the critical mechanism underlying Ad-p53-mediated apoptosis and may explain the resistance of gliomas to conventional therapy. Thus, the hypothesis of this proposal is that site-specific phosphorylation of p53 regulates the induction of Ad-p53-mediated apoptosis in glioma cells, and that p53 phosphorylation can be modulated with DNA-damaging agents to overcome the resistance of wild-type p53 gliomas to conventional therapy. To test this hypothesis we will construct p53 expression vectors in which critical N-terminal serines of p53 are mutated either to prevent their phosphorylation or to mimic constitution phosphorylation. By analyzing the apoptotic effects of these constructs on mutant and wildtype p53 gliomas with or without DNA damaging agents as well as in isogenic cell lines we will characterize the N-terminal phosphorylation sites that are critically involved in regulating p53-mediated apoptosis in glioma cells (Aim 1). By analyzing the expression of p53-responsive down-stream targets after transfer of the N-terminal serine mutant constructs into glioma cells, we will determine whether p53 phosphorylation regulates apoptosis by altering the spectrum of p53 responsive genes (Aim 2). To investigate the effects of p53 phosphorylation on Ad-p53-mediated apoptosis in vivo, human xenografts grown intracranially in nude mice will be treated with Ad-p53 with or without radiation or chemotherapy and levels of p53 phosphorylation will be correlated with apoptosis and animal survival. These studies should help define p53 phosphorylation as a therapeutic target and a marker of p53-mediated apoptosis in gliomas.